The Research Plan is directed at understanding regulation of the calcium-activated potassium channel by the cell. This large-conductance ion channel is ubiquitous and appears to play several roles in maintaining cell homeostasis. Two complementary approaches to studying regulation are used, each employing patch clamp techniques for recording from either single Ca2+-activated K+ channels, or macroscopic currents from the entire cell. The Ca2+ dependence of channel inactivation will be studied in excised patches of mambrane from rat skeletel muscle cell culture. Inactivation will be measured from averaged single channel currents which result from depolarizing voltage steps, and from the distribution of steady-state shut intervals. This study extends previous work from this lab by increasing our knowledge of the Ca2+ dependence of this channel, with particular regard for the detection of inactivation at physiological concentrations of calcium. The second approach exploits previous work and employes the Ca2+-activated K+ channel as a specific and sensitive intracellular Ca2+ probe. Using the 1321N1 human astrocytoma cell line, channel activity from cell-attached membrane patches will be used to monitor the changes in intracellular Ca2+ which apparently accompany muscarinic receptor activation. The astrocytoma cells have served in the past as a model biochemical system for the study of the relation between muscarinic receptor activation and the intracellular second messengers cAMP and calcium. Competitive antagonists such as atropine will be used to determine whether the changes in channel activity following bath- or pressure-application of carbachol resulted from muscarinic receptor activation. The assumption that Ca2+ alone is affecting channel activity will be tested by introducing EGTA in the the cell interior, and by exposing excised patches to known intermediates of the receptor-Ca2+ linkage. The origin of the mobilized Ca2+ will also be determined by testing for the cholinergic response in the presence and absence of extracellular Ca2+. As in many other cell types, Ca2+ mobilization in the astrocytoma cell accompanies phosphoinositide breakdown. The ability to quantitate changes in intracellular calcium will provide an important tool for studying this important regulatory and signalling mechanism.